Feb 14, 2022
MDA for virome analysis
- 1Copenhagen University
- FOOD Micro UCPH
Protocol Citation: Frej Larsen 2022. MDA for virome analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.b42uqyew
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 14, 2022
Last Modified: February 14, 2022
Protocol Integer ID: 58164
Abstract
This protocol is not intended for amplification of DNA. Rather, it uses the Phi29 DNA polymerase enzyme to turn single stranded DNA (ssDNA) into double stranded DNA (dsDNA). This is required for ssDNA viruses to be sequenced.
The procedure should be performed in a fume hood with a UV-light. Prior to starting, the UV-light should be turned on to disinfect the workspace and UV-tolerant materials such as empty PCR-plates, lids, and pipette tips.
Whenever working with the samples, keep them on ice.
Place materials that tolerate UV-treatment in fume hood and turn on the UV light and recirculation for 30 minutes before use.
Pick up a bucket of ice and thaw samples on ice
Add 10 µL denaturation buffer to a PCR tube
Add 10 ng DNA in 10 µL MQ water to the PCR tube and mix by pipetting.
If sample has a concentration below 1 ng/ul, add 10 µL of undiluted sample to the tube and mix by pipetting
Briefly centrifuge the plate
Denature template DNA by 95 degrees for 3 minutes in a ThermoCycler. Directly after, put the samples back on ice
Add all 20 µL sample content to the MDA cake tube. Keep both sample and cake on ice while transfering the sample
Seal tube with provided lid and briefly centrifuge the sample
Run sample on a ThermoCycler with the following program:
- 30 °C for 00:30:00
- 65 °C for 00:10:00
- 4 °C
40m
Purify samples with bead purification or store sample at -20 °C or -80 °C